WorkShop For Medical Officers

WORKSHOP FOR
MEDICAL OFFICERS

Reading Material

Dr. S.B.Upadhyay
Director

Forensic Science Laboratory, UP

Mahanagar, Lucknow – 226006
Phone/Fax – 0522-2336232
[email protected]

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Table of Contents

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A. Collection of Evidence for DNA Fingerprinting …… 2-40
…………………………………………………..Annexure

B. Viscera Management System ………………………….. 1-32
…………………………………………….Annexure

C. Wound Ballistics ……………………………………….. 1-6

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Collection of Evidence for DNA Fingerprinting

Crime is as old as human civilization. The primary aim of forensic

examination is to collect the evidence that may help to prove or disprove a link
between individuals and / or between individuals and objects or places. DNA ie.
Deoxyribonucleic acid is the vehicle of generational transference of heritable unit.

Forensic DNA analysis or DNA Fingerprinting helps in establishing an
association between biological evidence and its source ie a suspect,
victim, crime scene or weapon . In analysis, specific genes in the
questioned item are compared with those in the known specimen.

Collection, preservation and handling are the integral part of DNA
fingerprinting.“Medico legal” term incorporates the basis of two sister professions
ie medicine and law. The medicolegal experts can provide a link between these
two professions for smooth effective functioning in a scientific manner. Area
where medicolegal experts may have to collect specimens for DNA fingerprinting
are-

• Paternity disputes
• Sexual offences
• Heinous crime
• Mass Disaster

Earlier forensic scientists have been employing gene coded
polymorphic products to link a suspect to the crime . They have been
using different techniques like blood grouping, HLA typing, isozyme
grouping etc. in the biological material recovered at the scene of crime

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for investigation. These tests are based on proteins, which get highly
degraded with time. Thus there was necessity for identification of
biological material, which on the one hand is very stable and on the
other hand is so variable that it is individual specific. The discovery of
DNA fingerprinting caught the imagination of forensic scientists to link
with certainty the origin of biological material.

DNA fingerprinting has had a major impact on the criminal
justice system and law during the last decade of the 20 th century. It has
been employed in criminal law to help prove guilt or innocence , in
family law to prove paternity , and in immigration law to prove blood
relationships or to establish citizenship. Its usefulness as a human
identification tool is evident. Accordingly in recent years our legal
system has given DNA fingerprinting the credibility that nature has
given it as the blue print of life.

WHAT IS DNA?

DNA or deoxyribonucleic acid is the genetic material and the
carrier of genetic traits. A part of DNA of various individuals is the
same, but another part differs from individual to individual (except in
the case of identical twins). Portions of DNA structure of certain genes
are as unique to each individual as fingerprints. Alec Jeffreys and his
colleagues who were responsible for these revelations named the process
for isolating and reading these DNA markers as "DNA Fingerprinting".
In forensic examination narrowing the source of biological material i.e.

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individualization remains an elusive goal. DNA fingerprinting has
brought forensic scientists to the brink of this goal. It provides a virtually
foolproof method of establishing identity including parentage of an
individual.

The human body is made up of 60 trillion cells. In cell are strands
of genetic material called chromosomes. Each human cell (except red.
blood cell) contains 23 pairs of chromosomes-23 from the father and 23
from the mother. Each chromosome consists of a long chemical
molecule of DNA complexed with proteins. DNA is a polymer ie, a
chain of small repeated sub units found in the nuclei of all human cells
(except red blood cells) and sub cellular structure mitochondria. The
structural unit of DNA is deoxyribonucleotide. A segment of DNA ,
which instructs the body cells to make proteins that determine
everything from eye colour to our susceptibility to resist diseases is the
fundamental unit of heredity called gene. However function of 95
percent of DNA is not yet understood and is known as junk DNA. In
which lies the mystery of individualization, ie, why one person is so
different from another. DNA fingerprints of different unrelated
individuals are different while related individuals show a high
coefficient of similarity.

DNA is made up of three components- (i) nitrogen bases (ii)
carbohydrates (deoxyribose sugar) and (iii) phosphate. There are four
types of nitrogen containing bases in DNA—Adenine(A), Guanine(G),

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Thymine(T) & Cytosine(C). Adenine and Guanine are called purine and
Thymine and Cytosine are pyrimidine. Sugar along with phosphate
groups form the backbone to which these bases are linked.

The double helix model of DNA was proposed by Watson and
Crick (1953). The two strands are linked by hydrogen bonds between
pair of base pairs. Adenine on one strand always pairs with thymine via
two hydrogen bonds (A=T) and guanine with cytosine via three
hydrogen bonds (G≡C). Out of 3.3 billion base pairs that make a human
being, approximately 3 million differ between any two individuals ie
only one – tenth of a single percent of DNA differs from one person to
the other.

DNA FINGERPRINTING
DNA fingerprinting or DNA typing or DNA profiling is a

technique that detects DNA pattern unique for every individual. It is a
complex process of analysis of some highly variable regions of DNA. It
involves the intersection of several scientific disciplines, including
molecular biology, genetics and statistical analysis. It is a reliable
molecular tool for the law agencies for resolving certain critical issues in
crime investigation.

Portions of the DNA molecule of the non coding region contain
sequences of letters that are repeated numerous times (tandem repeats)
and offer a means of distinguishing one individual from another through

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DNA fingerprinting. Within the world's population there are numerous
possibilities for the number of times a particular sequence of base letters
can repeat themselves on a DNA strand. The stuttered region of DNA
where a short sequence of bases, typically 20 base pair long, is repeated
over and over again and is called "minisatellite”. In case of
"microsatellites" a short sequence of DNA, about 3 to 7 base pair long,
is repeated. Forensic scientists scan 15 different regions that vary from
person to person and used the data to create a DNA profile / fingerprint
of that individual. The probability that another person has the same
DNA profile for a particular set of regions is extremely low. In criminal
cases the scientists extract the DNA from different samples and analyze
it for the presence of a set of specific DNA regions (markers). The
following DNA technologies are used in forensic investigations –

(i)Restriction Fragment Length Polymorphism (RFLP)

The methods used in DNA fingerprinting are conventional
techniques of molecular biology. The first technique that was adopted
for forensic DNA analysis was RFLP.

Unfortunately, the RFLP technique requires a greater amount of
better quality DNA than the newer PCR based techniques. In addition
forensic evidence is often old, degraded and of limited quantity where
RFLP is sometimes not possible.

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(ii)PCR Analysis

Polymerase Chain Reaction (PCR) is used to make millions of
exact copies of DNA from biological samples. DNA amplification with
PCR makes possible DNA analysis on biological samples as small as
few skin cells.

(iii) STR Analysis

The latest method of DNA fingerprinting, short tandem repeat
(STR) or microsatellite analysis has the potential for a higher
discrimination and also reduces the amount of time to obtain results. It
also requires a sample size smaller than that needed for RFLP methods.
STR technology is used to evaluate specific regions (loci) within nuclear
DNA. STRs are locations (loci) on chromosome that contain short
sequence elements that repeat themselves within the DNA molecule.
The repeat sequence as mentioned earlier is 3-7 bases and the entire
strand of STR less than 400 bases in length. Hence, STRs are less
susceptible to degradation and may often be recovered from bodies or
stains that have been subjected to extreme decomposition. The Federal
Bureau of Investigation (FBI) uses a standard set of thirteen specific
STR regions. The odds that the two individuals will have the same 13
loci DNA profile is about one in one billion. The following steps are
involved in STR analysis -

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• Extracting and purifying DNA from biological evidence.
• Amplification of selected genetic markers through PCR ie

polymerase chain reaction.
• Visualizing the fragments and genotyping.
• Statistical analysis and Interpretation.

(IV) Mitochondrial DNA analysis (mt DNA)
In the investigation of cases that have gone unsolved for many

years mt DNA is extremely valuable . Nuclear DNA must be extracted
from samples for RFLP, PCR and STR, however mt DNA analysis uses
DNA extracted from mitochondria (another cellular organelle). Older
biological samples that lack nucleated cellular material for example hair,
bones and teeth can be analysed with mt DNA. All daughters have the
same mt DNA as their mothers because mitochondria of each embryo
comes from mother’s egg cell- father’s sperm contributes only nuclear
DNA. Comparison of mt DNA profile with profile of a potential
maternal relative can be an important technique in solving missing
persons identity and maternity disputes.

(V) Y-chromosome Analysis
The Y-chromosome is passed directly from father to son and hence

the analysis of genetic markers of Y-chromosome is especially useful for
analyzing biological evidence involving multiple male contributors.

The advantages of DNA Fingerprinting can be summarized as
follows -

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• Discrimination potential is very high hence individuals as close as
brothers or sisters (except monozygotic twins) can be identified.

• DNA profiling is feasible even from degraded or very minute amount
of biological material (at times invisible to the naked eye) because of
high sensitivity.

• DNA molecule is very stable.
• DNA profiling can be done from any biological material and is not

restricted to any specific organ/area of the body, unlike dermal
fingerprinting.
• Determination of species of origin and gender is feasible.
• DNA fingerprinting leads to better administration of justice and
increased public confidence in the Criminal Justice System.
APPLICATIONS
(A) Civil Cases
1. In proving paternity/maternity
2. Solving cases of switched babies
3. Determining immigration status
4. Identification of victims of accident, fire, natural disasters etc.
5. Delineating family lineage

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(B) Criminal Cases
1. Solving murder cases
2. Linking victim and culprit in sexual offences
3. Identification of mutilated bodies/ Skeletons/ source of tooth pulp
4. Sexing biological material
5. Solving crimes related to animals
6. Solving crimes related to plants

LEGISLATIONS

Cr.P.C and DNA Testing

(i) Section 53, Cr.P.C.-
The title or marginal notes of section 53 is “Examination of

accused by medical practitioner at the request of police officer”.
No doubt the provisions of section 53 are applicable during
investigation and when a person is in custody after his arrest on a
charge. As per the section -

1. When a person is arrested on a charge of committing an
offence of such a nature and alleged to have been
committed under such circumstances that there are
reasonable grounds for belonging that an examination of
his person will afford evidence as to the commission of an
offence, it shall be lawful for registered medical

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practitioner, acting at the request of a police officer not
below the rank of sub inspector, and for any person acting
in good faith in his aid and under his direction, to make
such an examination of a person arrested as is reasonably,
necessary in order to ascertain the facts which may afford
the such evidence, and to use such force as is reasonably
necessary for that purpose.
2. Whenever the person of a female is to be examined under
this section, the examination shall be made only by, or
under the supervision of, a female registered medical
practitioner.
[Explanation- In this section and in sections 53-A and 54,-

(a) “examination” shall include the examination of blood, blood
stains, semen, swabs in case of sexual offence, sputum and
sweat, hair samples and fingernail clipping by the use of
modern and scientific techniques including DNA profiling and
such other tests which the registered medical practitioner
thinks necessary in a particular case;

(b) “registered medical practitioner” means a medical practitioner
who possess any medical qualification as defined in cause (h)
of section 2 of the Indian Medical Council Act 1956 and whose
name has been entered in a State Medical Register.]

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(ii) Section 53A, Cr.P.C.-
Section 53A relates to specific accused who has been arrested for
charges of rape or its attempt and there are reasonable grounds for
believing that the examination of his person would afford evidence, the
accused may be examined medically at his arrest and even a reasonable
force may be used, which is necessary for the purpose for such
examination. The examination of the accused has been subjected to the
condition, that “there are reasonable grounds for believing” that
examination would afford evidence.

(iii) Section 164A, Cr.P.C.-
The provision of section 164A relates to examination of a victim of
rape, including taking a sample from her person, with her consent for
“DNA profiling”. For a very long time victims of any kind of assault and
injury, including victims of rape had been medically and forensically
examined and the examination report prepared during such examinations
was produced in courts as evidence.

As per the section, the registered medical practitioner, to whom
such woman is sent shall, without delay, examine her person and prepare
a report of his examination giving the following particulars, namely-

(i) the name and the address of woman and of the person by
whom she was brought;

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(ii) the age of the woman;

(iii) the description of the material taken from the person of the
woman for DNA profiling;

(iv) marks of injury, if any, on the person of the woman;

(v) general mental condition of the woman; and

(vi) other material particulars in reasonable details.

It seems that there is sufficient law ii existence to deal with DNA
evidence, in the state it is at present. Where peculiar situations arise the
courts have inherent powers to deal with such situations as they have
been doing now.

MEDICOLEGAL ASPECTS
A. MEDICO LEGAL ASPECTS OF SEXUAL OFFENCE

Collection of Forensic DNA Evidence

The rationale for collecting forensic evidence is to link a suspect to the
victim of the crime. In order to collect suitable forensic evidence, the health worker
must understand the types of evidence that may be present. The selection of the
sample taken should be directed, in part, by the hospital or clinic. Specimen
collection should be performed as soon as possible in order to minimize loss and
degradation of the sample (e.g. loss of semen from drainage, douching, etc.).
Control sample

A general principle when collecting evidence for forensic purposes is to
collect a control or reference sample at the same time as any evidence or test
sample, which may be connected to the crime. A control sample is a known
sample, for example, blood, hair or tissue, which may be compared with a sample

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obtained from the crime scene (e.g. semen from the vagina of a rape survivor or
blood-stained clothing).
Storage of samples and preservation of chain of evidence

Steps to ensure that the evidence is reliable include: using appropriate
containers or bags; proper labeling; proper sealing (e.g. use of tamper-proof seals);
secure storage; and maintenance of the chain of custody. This will result in the
forensic analyses being acceptable to the court. The samples should be initialed
and dated by the collector and handed to the investigating officer who should sign
for their receipt. If there is any detay in handing the samples to the investigating
officer or laboratory, the samples should be securely stored, preferably in locked
facility with restricted access until they are handed over.
Preservation of samples

Wet samples (e.g. clothing) should be air dried to prevent growth of bacteria
and fungi which may degrade DNA making the sample unsuitable for analysis.
Samples containing biological evidentiary material such as DNA should be stored
in a cool dry environment or refrigerated to prevent putrefaction (decomposition)
which may render the sample unsuitable for analysis. Care must be taken to avoid
contamination of the sample by the health worker who may inadvertently add
material such as hair, blood or bacteria during collection, preservation, or handling
of the sample.
Taking of swabs

The purpose of taking swabs is to collect samples of any body fluids that
may have been deposited on the outside of the rape survivor's body or on any
interior orifice which may assist to connect the assailant with the alleged assault
through DNA analysis of the samples. The areas of the body from which the
samples are taken may help to corroborate the survivor's version of the events that
are alleged to have occurred. These areas include the inside of the mouth (for

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evidence of semen) or the exterior of the vagina (for evidence of saliva) if oral sex
was performed; the areas around the aureole or breast if they were bitten, licked or
sucked by the assailant (for evidence of saliva); the face, neck, cheeks or lips of the
mouth if they were kissed or licked by the assailant (for evidence of saliva); any
areas of the body, head or limbs on which the assailant any have ejaculated (for
evidence of semen); and under the fingernails where the survivor may have
scratched the assailant.
Body fluids

Body fluids such as blood or semen are likely to remain stuck on the rape
survivor's skin if they become air-dried and are not washed off or absorbed by
materials such as clothing or bedding. Saliva may evaporate but will leave cells
from the oral mucosa behind. The length of time that spermatozoa will last in a
survivor will depend upon where it is deposited on the body.

B. MEDICOLEGAL WORK IN MASS DISASTER
There is no justification from the medico-legal standpoint not to follow all
scientific procedures for the recovery, transfer, identification, and final disposal of
the remains of disaster fatalities. A select group of experts who are experienced in
these procedures should oversee the process. However, in situations where experts
are not available, the community physician should take leadership and make use of
all available resources to carry out the job.

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DNA Identification

DNA analysis

Consideration of established
techniques

Choice of most informative
and valid technique

Application of DNA according to
necessity and availability of each

sample

Types of DNA

Nuclear DNA Mitochondrial DNA

DNA of autosomal DNA of sex
chromosomes chromosomes

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C. MEDICOLEGAL WORK IN PARENTAGE DISPUTE
Presently blood is the classic, conclusive sample for determining DNA. Blood

can be obtained by capillary or venous puncture.
The following table lists the range of forensic specimens that are typically of

interest for a medicolegal expert along with appropriate collection techniques and
comments on their relevance.

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Table 1.Forensic Specimens

Site Material Sampling Sampling Instruction

Anus Semen Cotton swabs and Use swab and slides to collect and
(rectum) microscope slide plate material; lubricate instruments
with water, not lubricant.
Lubricant Cotton swab
Cotton swab, dry swab after
Blood DNA (Victim) Appropriate tube collection.

Clothing Adherent foreign Paper bag(s) Collect 2 - 5 ml of venous blood in
materials EDTA vial or prepare stain on FTA
(e.g. semen, paper.
blood, hair,
fibers) . Clothing should be placed in a
paper bag(s). Collect paper sheet or
Genitalia Semen Cotton swabs and drop cloth. After air dry wet items
Male & microscope should be bagged separately.
female slide
Use separate swabs and slides to
Hair Comparison of Sterile container collect and plate material collected
Mouth hair found at from the external genitalia, vaginal
crime scene Cotton swabs, vault and cervix; lubricate speculum
Semen sterile container with water not lubricant or collect a
blind vaginal swab.

Cut approximately 20 hairs and
place hair in sterile container.

Swab multiple sites in mouth with
one or more swabs. To obtain a
sample of oral washing, rinse mouth
with 10 ml water and collect in
sterile container

DNA (victim) Cotton swabs

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Nails Skin, fibers, Sterile toothpick Use the toothpick to collect material
from under the nails or the nail(s)
blood etc. Or similar or nail can be cut and the clippings
collected in a sterile container.
(from assailant) scissors/clippers
Collect if used during or after
Sanitary Foreign material Sterile container vaginal or oral penetration.
pads/ (e.g. semen,
tampons blood, hair) Swab sites where semen may be
present.
Skin Semen Cotton swab Dry swab after collection.

Saliva (e.g. at Cotton swab Place material in sterile container
sites of kissing, (e.g. envelope, bottle)
biting or
licking), blood

Foreign material Swab or tweezers
(e.g. vegetation,
matted hair or
foreign hairs)

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Table 2. Collection of Forensic DNA Evidence

SPECIMENS COLLECTION PURPOSE
/ SAMPLES
• Air dry wet or blood stained • Identification of assailant
Clothing clothing (do not use extreme using semen, blood or
sources of heat e.g. blower, saliva stains or hair on
Semen hairdryer etc.) clothing

• Once dried, place in paper bags • To show corroborative
(not plastic) evidence of force used eg.
Torn clothing
• Do not store wet clothing.
• Ensure packaging is properly • To identify place where
the crime was committed
labeled and delivered to
laboratory as early as possible. • Identification of assailant
• If delivery is to be delayed, • Confirmation of samples
store in a secure cool dry place.
of semen
• Semen on clothing should be
treated as above. Collection of
semen from body surface,
mouth, anus, genitalia should
follow the protocol given
below-

• A smear on glass slide by
rolling a swab over a slide
with out rubbing it as the latter
may cause the spermatozoa to
break and thereby give a false
negative result. The slide is
then air dried.

• Fixative should not be used
• All samples should be

properly sealed, packaged and
labeled.

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Blood • Prepare stain on FTA paper; or • To ascertain if the sample
Pubic or
Head hair • At least 2-5 ml blood should be is from assailant or
collected as a control sample in survivor, is with consent
Bones
EDTA vial. to intercourse/ defend her

• Blood- stained clothing and self.

objects should be treated as

described above.

• Separate items that are blood

stained should be packaged

individually and labeled and

treated as above.

• Hair sample should be Identification of assailant vs.

collected using clean sterilized survivor.

forceps

• Individual hairs or clumps of

hair should be separately

packaged,

• Hair with root tissues, or mixed

with blood, body fluids or other

tissue must be carefully

collected to retain the integrity

of the samples.

• Hair mixed with wet fluids

should be air dried as described

above

• Control sample of body, scalp,

auxiliary and pubic hair should

be taken,

• Ideally by plucking and not by

cutting the tips to obtain hair

roots that contain adequate

DNA analysis.

• Wash with running water then Identification of body.

air dry, place material in sterile

cloth bound envelope/ wrap in

paper or cloth.

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Table 3- DNA Content of Tissues

Source DNA content (approximate)

Amniotic fluid 65 ng/ml (1X104 cells/ml at 16 weeks

Blood gestation)
40 µl (1 µl = 4 X103 to 11 X103 WBC, 1

WBC= 6.6 pg DNA

CVS 8 µg/mg

Hair roots 250 ng/plucked hair root

Liver 15 µg/mg

Muscle 3 µg/mg

Sperm 3.3 pg/cell

BASIC DO’s & DONT’s
Do’s:

GENERAL (WITH SEXUAL ASSAULT VICTIM)

• Make sure you understand your own attitudes and feeling about sexual
assault

• Let the victim know you believe her
• Let the victim know she survived, and that is not failure but success
• Encourage the victim cry, yell or talk
• Listen

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TECHNICAL---

• To establish identity of deceased from skeletal remains, always collect
intact long bones (femur, humerus)/ molar teeth in duplicate.

• Preserve tissue, foetus and other similar samples in 0.9% DNS and keep
it in refrigerator for a short period if there is any delay in forwarding the
sample to the laboratory.

• Always wrap stained clothes and fabrics in paper sheets and pack in
cotton cloth or aerated container.

• If there are more than one sample, pack them separately.
• From dead body always take two or more types of samples in duplicate.
• Submit samples in laboratory without any delay.
• Always use disposable gloves, mask, syringes, dropper, blade, scissors

for handling and collecting samples.
Don’ts:

GENERAL (WITH SEXUAL ASSAULT VICTIM)

• Do things for the victim without asking her first
• Get angry with victim
• Blame the victim
• Boss the victim around
• Rant and rave at the offender
• Try to make the victim believe the sexual assault was not serious

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TECHNICAL-

• Never prefer to collect the clavicle bone.
• Never use formalin to preserve tissue and bones.
• Do not pack clothes/ garments, stain and swabs in wet condition.
• Never dry stains, swabs in direct sunlight by using heater, hot air blower etc.
• Never use cork/lid in vials while packing swab of semen, blood and other

body fluids.
• Do not send completely burnt/ broken bones, burnt or singed hair.
• Never use polythene bag as packing material for biological evidence.

GUIDELINES
A. Evidence collection in sexual offences
Technical-
Step1: Oral specimen

Collect seminal fluid in the oral cavity for DNA analysis in cases where
there is suspected oro-genital contact.
Step2: Collection of panties and sanitary pad

Collect the panties worn by the survivor during or after the incident. The
sanitary pad must not be removed if it is attached to the panties. If the sanitary
pad was detached at the time of medical examination, the sticky side of sanitary
pad must be covered by waxed sheet to prevent the pad from sticking to the
paper collection bag.

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Step 3: Evidence of the patient’s body
Collect and preserve any physical or biological evidence that may be present

on patient’s hair, skin or fingernails and to collect additional foreign debris. The
site to be sampled is determined by asking the patient, by examination and by
use of an ultraviolet light source, which will reveal stains that are invisible in
normal light. The swab should be placed in dry rack of the swab guard box. If
the bite marks are present they should be photographed after taking the
necessary swab.

Step 4: Pubic hair

Comb to obtain any loose hair or debris that may help in identifying the
assailant. Any matted hair may indicate the presence of blood or semen and
should be cut over another sheet of catch paper so that it falls on to the paper
which should also be folded and labeled.

For reference purposes in order to obtain a comparative sample from the
patient she should be asked to allow about 10 hairs to be pulled from her pubic
region.
Step5: Collection of ano-rectal specimens

Conduct a through examination of ano-rectal region in order to record
trauma and to obtain biological material for DNA analysis. In cases of possible
ano-rectal assault external and rectal swabs should be collected.
The patient should be placed in a comfortable position for an anal examination
and swabbing. The swab be slightly moistened with sterile water and the anus
carefully swabbed, slightly extending into the anal canal.

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Step 5: Genital specimens

Perform a through examination of the genital area in order to identify and
record any trauma and to obtain biological material for DNA analysis which will
assist in identifying the suspect.

• Swab of the external genitalia in order to collect any saliva or semen
that may be present Moisten the swab with sterile water and swab the external and
internal surfaces of labia majora, including the clitoris, the periurethral area and the
fossa navicularis.

• Take a swab of interior and posterior vaginal fornices using a
speculum before an internal digital examination is performed.

• Swab the cervix for collecting as much of mucous plug as possible.
Others:
(1) Contamination: Wear gloves at all times both to protect yourself and to
avoid contamination of evidence.

Be aware that DNA contamination can occur easily—
Avoid coughing or sneezing over swabs and ensure that any surfaces used for
swabs are uncontaminated. Use disposable paper towels on surfaces.
(II) Reference sample: Sample provided by a known person, for instance a
victim or suspect for DNA analysis are reference samples.
Forensic DNA analysis is a science of comparison and reference blood samples
of both victim and suspects are required for comparison of profiles generated from
evidence material.
(III) Swabs and slides for trace evidence: Sterile swab to be used for the
recovery of biological samples from individuals and crime scenes; ensure that these
items are guaranteed to be sterile and uncontaminated, with a seal that you can break
yourself.

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Prepare dry mounts by smearing each swab onto appropriate microscope slide.
Allow the smear to air dry, label with examinee’s name, date and indicate which
swab the slide was made from.

Types of swabs collected for the purpose of a reference DNA samples
Buccal or oral swab (saliva/ bite marks), vaginal and cervix swabs (semen),
penial swabs etc.
Label each specimen on the envelopes as follows:

• Name of examine
• Date of examination, time of examination
• Name of individual sample
• Site of collection if applicable
• Name of examining doctor
• Signature of examining doctor on seal of envelops etc.
• FIR No. and Parcel No.
• Description of specimen seal
• Name of the accused/victim

(IV) Fingernail scrapings and trace evidence

Fingernail clippings and fingernail scrapings from the left and right hands
should be collected in pre-labeled self-sealing plastic bags and put into labeled
envelope.

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(V) Hair samples

• Different combs should be used to collect any loose hair or fibres from
the head and pubic area over the piece of clean paper. The pubic hair
combing and the comb are placed in the envelope.

• Where there is evidence of semen or other matted materials on pubic or
head hair, it may be collected with the help of a moistened swab. The
swab should be placed in a small paper envelope and labeled “Possible
secretion sample from head (pubic) hair.

The second approach is DNA analysis of the hair root and/or sheath of the root.
However, the sheath cells surrounding hair roots are more likely to be present
when the hair is pulled from the scalp, as might happen, for example during
assault/violence.

B. Evidence collection in autopsy samples

Tissue and aborted foetus
• Deep muscle tissue samples and aborted foetus for DNA analysis
should be collected in plastic bottles and transported on dry ice.

Bone
• Preferably long bones (femur/humerus) and sternum should be sent
for DNA analysis. Totally charred bones should not be sent for DNA
profiling.
• Exhumed bones should be cleaned properly. All the sticking debris
should be removed.
• Clean bone should be packaged.

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C. Evidence collection from clothing

a. To minimize loss of evidence a hospital sheet should be placed on the
floor and a clean paper sheet should be placed on the top of the sheet.
The patient should disrobe over the paper sheet.

b. After air drying items such dresses, blouses, shirts etc. should be put
into paper bags.

c. Any wet stains, such as blood or semen should be allowed to air dry
before being place into paper bags. It is preferable that each piece of
clothing be folded inward, placing a piece of clean paper against any
stain, so that the stains are not in contact with the bag or other part of
the clothing.

D. Evidence collection of reference samples

Collection of reference samples

Source of reference DNA
for forensic identification

Reference DNA

Personal effects of Relatives of unidentified
unidentified individuals individuals

For secondary Use Principal reference DNA

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(I) Conclusive samples from living subjects
• Blood
Blood is the classic, conclusive sample for determining DNA. Blood can be

obtained by capillary or venous puncture.
• Buccal epithelial cells
These cells are collected from the inside of the subject’s cheeks, using sterile

dry swabs. Two samples are taken- one swab is rubbed on the inside of the left
cheek and another swab is used on the right cheek. The swab should be identified
and left to dry at room temperature in a protected area. They must not be placed in
a container until they are completely dry since the bacteria in saliva proliferate
rapidly in moist conditions and will degrade DNA.

• Hair follicles
Between 10 and 15 hairs with roots should be pulled from the subject.

Source of DNA from
Victims

Primary tissues

Soft tissue Bone tissue Bone marrow

Muscle tissue

Skeletal tissue

Cardiac tissue

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(II) Conclusive samples in dead bodies
(a) Conclusive samples in well preserved bodies

• Post mortem blood.
A sample of about 10 ml of blood should be drawn into a tube containing

an anticoagulant (EDTA type).
• Skeletal muscle.

Select two skeletal muscle fragments (weighing about 10g and
approximately 2 cm wide) from the best preserved area of the body, and
place them in a plastic container that has a wide mouth and screw-on lid.
This type of tissue is preferable because along with cardiac muscle, it is the
most resistant to decomposition.
• Teeth.
If there are doubts about the preservation of the corpse, it is advisable to
extract four teeth, preferably molars and save them so that exhumation of the body
for identification purposes can be avoided. Prior to the extraction, a dental chart
should be completed.
(b) Conclusive samples in charred corpses
Despite the external appearance, the stability of DNA at high temperatures
allows genetic analysis in corpses where charring is not complete by using
fragments of skeletal from deep regions of the body, and from semi-solid blood
that remains inside cardiac cavities. If charring is total, it is advisable to contact the
laboratory for an evaluation of the available samples and their condition to
determine which would be most appropriate for analysis.

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(c) Conclusive samples in decomposed or skeletonized corpses
Remaining decomposed tissue should be removed from bone, and a long bone,

preferably the femur, should be used. If not possible to obtain this sample, the
laboratory should evaluate available samples and their condition to determine
which would be most appropriate for analysis.

• Teeth
After a dental chart has been completed, select at least four teeth, molars
where possible. The samples should not have been damaged or subjected to
endodontia.
E. Preventive Guidelines

i. Personnel Protection Guidelines
All body fluids should be regarded as potentially infective.

• Cover any cuts or graze on hands with waterproof dressings.
• Wash hands especially when beginning or ending a new task, before break

or meal times, before smoking, and at the beginning and end of duty periods.
ii. Disinfection Guidelines

• Commercial thick bleach can be used for spillages of biologically hazardous
materials. This should be left in contact with the contaminated area before
rinsing and wiping dry.

• For general disinfection e.g. work surfaces after handling biological
specimens, 1 in 10 dilution of commercial thick bleach should be used as
above. It should be noted that dilution of thick bleach do not remain
effective for periods in excess of a few days.

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iii. Taking DNA Reference Samples
• The person taking samples must wear gloves throughout the whole sampling

procedure.
• Open the sampling kit and assure that the kit is complete checking off each

item against the checklist provided. Follow the sampling instruction.
• If at any time during the sampling process the sample taken is dropped or

comes into contact with any other surface the procedure should stop and the
sampling kit disposed of. Sample will then be taken using a new DNA
sampling kit.
• Once the samples have been successfully taken collect up the wrappers and
gloves and dispose of these using designated receptacles.
• Insert the details of the donor and other necessary information on the form
provided.
• Place the form together with the sample in the tamper evident container,
store and send to the laboratory as per legal instructions.

iv. Anti contamination
• Due to sensitivity of current DNA techniques extreme caution including

wearing of a face mask must be taken.
• All containers used for transportation e.g. cool boxes crates, boxes should be

cleaned prior to and after use.
• Wherever possible sterile disposables sampling materials should be used.
• Disposable gloves must always be worn over top cuffs and should be

changed after handling individual item/objects. Barrier clothing should also
be used as often as possible.
• For serious offences wear disposable face masks overshoes and suits fully
done up the hood up.

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• Handle items as little as possible and not re-open items for interview
purposes-use paper bags with transparent panels.

• Always handle one item at a time.
• Where possible take the container to the evidence and not the evidence to the

container.
• Contact between victim and suspect samples should be avoided at all times.
• Ensure that any person attending a crime scene has no contact with a suspect

or his/her clothing.
• Multiple suspects, the victim and their clothing must be kept apart at all

times and should not be allowed to come into contact with the same objects
Each item should be packaged sealed and labeled as soon as it is taken.
• Never pack several items/objects together.
• Use bags of a suitable size or shape, do not force items into packaging that
are too small, bags may tear or lids may be forced off.
• Seal all packaging securely; use adhesive tape on all edges.
• Never reuse packaging.
• If an item will not fit or packaging is used in error do not use it for a
different item. It must be discarded.
• Never eat drink or smoke when recovering evidential samples.
• Dry sample should be kept at room temperature (cool if possible) and out of
direct sunlight. Dry sample stored at ambient temperature should not
deteriorate/decompose/degrade and will remain suitable for future DNA
analysis. Breathable bags, cardboard packaging and brown bags will allow
samples to dry out whilst safely packed away and should be stored as above.

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• If samples are air dried then this must take place in an area free from any
contaminant for example in a sterile drying cabinet. If this is not achievable
and there is any risk of minor contamination then samples not be air dried.

• If samples are frozen then they should be kept frozen and never be allowed
to thaw and or refreeze since this will cause the break down of DNA.

• Plastic bags can on rare occasions be used to transport very wet items but
this should be on the instruction of the local forensic Science Laboratory.

• All sample containing biological materials should be placed into suitable
secondary packaging for transport to the laboratory. Local transportation
regulations should be adhered to- international biohazard sign can be used.

F. Precautions During Collection and dispatch of samples
(I) Protection of personnel

Prevent, at all times, direct contact by the worker with the sample, using
gloves, masks, gowns, or other protective clothing; Prohibit the consumption of food,
drink and tobacco products while handling the sample; Maximize asepsis and use
disposable materials whenever possible. Once sample collection is complete, places
all used disposable materials in containers for biological waste, and follow standards
for disposal of biological waste. When sample collection takes place in the autopsy
station, extreme precautions should be taken.
(II) Protection of samples

• Contamination by human biological material.
This occurs when human biological material is deposited at the site of the

event or in the corpse following the event. It can be caused by onlookers, family
members, or persons involved in the investigation who accidentally or out of
ignorance, contaminate the sample. This occurs frequently when minimal
precautions when collecting evidence are overlooked or packaging is defective.

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• Contamination or loss during transfer or biological evidence.
This occurs, usually accidentally, during the transfer of evidence from one site

to another and can result in the contamination or loss of a sample. It happens most
frequently when hair samples are moved.

• Microbiological contamination.
This type of contamination occurs when microorganisms develop, possibly as

a result of humidity or high temperatures. Normally the microorganisms grow or
proliferate because of defects in packaging or shortage prior to sending the samples
to a laboratory.

• Chemical contamination.
This makes it difficult to amplify and extract DNA. It occurs when samples

are immersed in preservatives such as formalin or when chemicals have been used in
previous tests (for example, fingerprints), thereby compromising DNA analysis.

• Systems for packing and preserving samples
Recommended packing and shipping procedures are outlined below:

1. Identification of the samples:
There should be enough space on all of the receptacles to identify the samples

and to write the following:
• Reference number of the sample;
• Type of sample:
• Ownership of sample, and location.

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2. Chain of custody.
There also should be a space dedicated to the chain of custody with the name

and signature of the person who collected the evidence, and the date and hour of
collection.
(III) Packaging
(a) Jars or receptacles

With liquid evidence, organs, soft tissue, etc. These receptacles should have
screw-on lids or airtight closures; they should already have been sealed with tape
and correctly identified, and should be kept refrigerated and sent to the laboratory
under refrigeration as soon as possible.
(b) Dry, Sterile swabs.

Swabs used to collect samples will be packed in small cardboard boxes
commercially designed for this purpose. This type of box protects the swabs and
allows them to completely dry out. Once identified. They will be sealed with tape
and sent without refrigeration to the laboratory. If it is not possible to obtain
specially designed boxes, once the swabs have been used to collect the biological
specimen they should be identified and numbered, placed in a protected area, and
allowed to dry completely at room temperature before being placed in a shipping
container. Once dry the swabs can be placed in correctly identified container,
sealed with tape, and sent to be laboratory.
(c) Samples with dry stains.

Each sample is placed on top of paper that will be folded and placed in a
paper bag, sealed with tape and correctly identified. This should be sent to the
laboratory without refrigeration.

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(d) Hairs, nails etc.
This kind of material should be collected in small pieces of paper that will

be carefully folded and put in a paper bag, sealed with tape, and correctly
identified. This should be sent to the laboratory without refrigeration.
(e) Bones and teeth.

These should be placed in a paper bags and cardboard boxes that are sealed
with tape and correctly identified. They can be sent to the laboratory without
refrigeration. If tissue is still attached to bones, airtight, plastic receptacles should
be sent to the laboratory as soon as possible.
INSTRUCTIONS

• In cases of maternity / paternity dispute blood samples of mother child and
suspected biological father are required.

• For identification of deceased/ unidentified corpses/ human remains, blood
samples of nearest relatives ie. Father, mother, children, brother, sister are
required along with the remains.

• While collecting sample from the dead body, at least two different types of
samples are collected – always collect samples in duplicate.

• The samples should be collected in following order of preference-
I. 2 to 5 ml blood collected directly from the heart (cardiac puncture)
II. In cases where no liquid blood can be obtained skeletal red muscles
(50-100 gms) should be collected in DNS.
III. Intact long bones in following order of preference-
(i) Femur
(ii) Tibia
(iii) Humerus
(iv) Teeth ( Preferably Molar)
(v) Ribs

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IV. In sexual assault cases exhibits collected from the victim/ suspect (garments,
swab, slide etc) alongwith blood samples of the victim/suspect and of suspect are
required.
SAMPLE COLLECTION KITS/ MATERIAL

(1) EDTA vial for whole blood
(2) FTA classic/ indicating card
(3) Buccal DNA collector
(4) Secur swab

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